Project Summary/Abstract Neurons communicate by releasing neuronal transmitters into the synaptic gap, and disruptions in the regulation of the release process can cause severe neurological and mental health disorders, such as Alzheimer's, schizophrenia, and epilepsy. Neurotransmitters are packed into synaptic vesicles and released via vesicle fusion with the presynaptic membrane. The release occurs at morphological specializations termed active zones (AZs). The release of neuronal transmitters is stochastic, and this property is thought to be fundamental for the information transfer. However the rules that govern the release of neuronal transmitters from individual AZs are not yet understood. We propose to take advantage of genetically encoded calcium indicator (GCaMP5) postsynaptically tagged to AZs at Drosophila neuromuscular junction. This marker enables optical monitoring of single vesicle fusion events at individual AZs. Release events can occur in response to a stimulus or spontaneously, and the spontaneous release component is critical for normal neuronal development and homeostasis. Extensive evidence suggests that the spontaneous release can be regulated by presynaptic Ca2+ transients, however it is still debated how this regulation occurs. To address this question we propose to combine optical and electrophysiology detection of spontaneous release events and thus to couple the spatial resolution of microscopy with the temporal resolution of electrophysiology. Our preliminary data suggests that spontaneous release at individual AZs does not occur randomly, but that its probability can be transiently elevated at specific AZs. In the present application we propose to investigate what factors determine this non-uniformity in the release probabilities. Specifically, we propose to investigate the impact of different presynaptic calcium sources, including the endoplasmic reticulum, mitochondria and spontaneous openings of voltage gated calcium channels. We propose to employ selective genetic and pharmacological manipulations of calcium transients from different sources and to investigate how this manipulations would affect the spatial and temporal non-uniformities in release probabilities at individual AZs. These approach will elucidate the fundamental mechanisms of the regulation of the spontaneous release component.